Relationship between blood manganese and bone mineral density and bone mineral content in adults: A population-based cross-sectional study

Purpose It has been reported that bone is the primary organ for manganese (Mn) accumulation, but the association between manganese and bone loss remains debatable. Therefore, this study aimed to evaluate the relationship between blood manganese and bone mineral density/bone mineral content (BMD/BMC) by using a representative sample from the National Health and Nutrition Examination Survey (NHANES). Methods A total of 9732 subjects over the age of 18 with available data were enrolled in this study. The relationship between blood manganese and BMD/BMC of the total body, spine and femoral regions was evaluated using multivariate linear regression models. Subgroup analyses were also performed. Results We observed a negative association between blood manganese and BMD/BMC in the femoral neck and total body in the fully adjusted model, especially femoral neck BMD in women aged 50–70 years. Conclusion In brief, people exposed to manganese should be aware of the increased risk of osteopenia or osteoporosis. Besides, due to the lack of available data, there are no definite values for the tolerable upper intake level (UL), average requirement (AR) and population reference intake (PRI) of manganese. The results of our study may provide some references for the establishment of AR, PRI and UL of Mn.

Introduction evaluate the relationship between blood manganese and BMD/BMC by using a representative sample from the National Health and Nutrition Examination Survey. A better understanding of the relationship between blood manganese and BMD may help to better identify patients with osteoporosis and prevent osteoporotic fractures.

Study design and population
The National Health and Nutrition Examination Survey (NHANES), a research program to assess the health and nutrition of adults and children in the United States, examining a nationally representative sample of approximately 5,000 people each year. In this study, we used NHANES data for 2013-2014 and 2017-2018. Of 19429 subjects, 12728 subjects with available blood manganese data were selected. After that, 2996 subjects lacking available Dual Energy Xray Absorptiometry (DEXA) data were excluded. Finally, A total of 9732 subjects with available DEXA data on total body (n = 3682), femur (n = 3752), and spine (n = 2298) were enrolled. The Ethics Review Committee of the National Center for Health Statistics approved all NHANES protocols and obtained written informed consents from all participants [16]. The flow chart (Fig 1) also shows the selection process. All methods were performed in accordance with the relevant guidelines and regulations.

Variables
The exposure variable was blood manganese, which was measured using mass spectrometry after a simple dilution sample preparation step. The Hologic Discovery A is a fan beam X-ray bone densitometer used to perform DXA examinations and estimate bone mineral content (BMC) and bone mineral density (BMD). DXA total-body scans will be performed on all individuals aged 18 to 59, while femur and spinal scans will be performed on samples aged 40 and older. Dependent variables were bone mineral density and bone mineral content of femur, lumbar spine and total body as measured by dual-energy X-ray absorptiometry. The following categorical variables were included as covariates into our analysis: gender, race/ethnicity (Mexican American, other Hispanic, non-Hispanic White, non-Hispanic Black, other races), drinking, hypertension, hypercholesterolemia, diabetes, difficulty walking or climbing stairs, renal insufficiency, asthma, arthritis, congestive heart failure, stroke, coronary heart disease, angina, thyroid problems, COPD, smoking, family smokers and osteoporosis. Our analysis included continuous covariates: age, body weight, standing height, and body mass index. Detailed information on blood manganese, BMD, BMC and covariates can be found on the NHANES website.

Statistical analysis
Data were analyzed using R software (version 3.6.1), IBM SPSS Statistics version 26 (IBM, Armonk, New York, USA) and GraphPad Prism version 8.3.0 (GraphPad Software, San Diego, California USA). Means with standard deviation (SD) were used for continuous characteristic variables and categorical variables were expressed as percentages or frequencies. Differences in categorical variables between exposed groups were analyzed by Pearson's chi-square tests. One-way ANOVA was used to analyze the differences of continuous variables among groups. Blood manganese levels were classified according to quartiles (quartile 1: < 25th percentile, quartile 2: 25th-50th percentile, quartile 3: 50th-75th percentile, quartile 4: > 75th percentile) and univariate and multivariable linear regression analysis were performed. In weighted univariate and multivariate linear regression models, we adjusted for potential confounders using an extended model approach to covariates to investigate the association between blood manganese levels and BMD/BMC (total femur, total spine, and total body). Including model 1 (unadjusted model), model 2 (adjusted for sex, age, race/ethnicity, and BMI), and model 3 (further adjusted for all relevant covariates in S1 Table). The results of regression analysis were represented by β regression coefficient, 95% confidence interval and P value, P < 0.05 (bilateral) was considered statistically significant.

Participant characteristics
9732 subjects with available total body (n = 3682), femur (n = 3752), and spine (n = 2298) DEXA data were enrolled in this study. The average whole blood manganese level is 10.38 ± 3.85 ug/L (range: 1.88 to 52.0 ug/L) in total body DEXA data, 9.64 ± 3.67 ug/L (range: 1.57 to 54.92 ug/L) in femur DEXA data, 9.81 ± 3.47 ug/L (range: 1.57 to 35.56 ug/L) in spine DEXA data. Classified based on quartiles, the weighted characteristics of these participants are shown in S1 Table. In the femur DEXA data group, the weighted mean age of the participants was 62.1±10.7 years, 48.9% (n = 1838) were female, and there were significant differences in baseline characteristics among the groups in age, sex, race, body mass index, alcohol consumption, hypertension, osteoarthritis, stroke, thyroid problems, smoking, and family smoking (all P < 0.05). In the spine DEXA data group, the weighted mean age of participants was 59.6±10.8 years, 54.6% (n = 1254) were female, and there were significant differences in baseline characteristics among the groups in age, sex, race, body mass index, alcohol consumption, hypertension, osteoarthritis, stroke, smoking status, diabetes, and family smoking (all P < 0.05). In the total body DEXA data group, the weighted mean age of participants was 37.7±12.4 years and 50.2% (n = 1848) were female. There were significant differences in baseline characteristics among the groups in age, sex, race, body mass index, hypercholesterolemia, thyroid problems, smoking, and family smoking.

Relationship between blood manganese levels and bone mineral density
Detailed results are represented in Tables 1-3 and S2 Table. Blood manganese levels are classified according to quartiles, the trends for blood manganese have statistical significance for left arm BMD (P for trend < 0.01); left leg BMD (P for trend < 0.01); right arm BMD (P for trend < 0.01); right leg BMD (P for trend < 0.01); left ribs BMD (P for trend < 0.01); thoracic spine BMD (P for trend < 0.05); lumber spine BMD (P for trend < 0.01); pelvis BMD (P for  trend < 0.01); trunk bone BMD (P for trend < 0.01); subtotal BMD (P for trend < 0.01); total BMD (P for trend < 0.01) in total body. In femur area the trends for blood manganese are statistically significant for total femur BMD (P for trend < 0.01); femoral neck BMD (P for trend < 0.05); intertrochanter BMD (P for trend < 0.01) and in lumbar spine area, the trends for blood manganese have statistical significance for total spine BMD (P for trend < 0.01); L1 BMD (P for trend < 0.05); L2 BMD (P for trend < 0.01); L3 BMD (P for trend < 0.01); L4 BMD (P for trend < 0.01).
Weighted univariate and multiple linear regression models are constructed in Tables 1-3. In the unadjusted model (Model 1), there is a significant negative correlation between increased blood manganese concentration and BMD of femur, spine and total body region

Relationship between blood manganese levels and bone mineral content
Detailed results are represented in Tables 1-3 and S2 Table. Blood manganese levels are classified according to quartiles, the trends for blood manganese have statistical significance  for all the BMC of femur, spine and total body region (all P for trend < 0.05). Weighted univariate and multiple linear regression models are constructed in Tables 1-3. In model 1, there was a significant negative correlation between increased blood manganese concentration and BMC of femur, spine and total body region [P<0.05 except for wards triangle BMC (β coefficient 0.0119; 95%CI -0.02, 0.0438) and head BMC (β coefficient -0.01685; 95%CI -0.0492, 0.0154)]. In model 3, BMC of femur, spine and total body, except wards triangle BMC (β coefficient -0.0268; 95%CI -0.0589, 0.0053; P 0.102), all decrease with increasing manganese levels.

Subgroup analysis
Subgroup analyses after adjusted for all relevant covariates are shown in Tables 4-6. Multiple regression analyses in the femoral region have showed that, stratified by sex, femoral neck BMD decreases with increased blood manganese (Femoral neck BMD: β coefficient -0.0544; 95%CI -0.0989, -0.0099; P 0.017), and femoral neck BMC decreases with increased blood manganese in all gender groups (β coefficient < 0, P < 0.05). Results stratified by race have showed that there are no significant correlation between femoral neck BMD/BMC and blood manganese in all races. The results of stratification by age have showed that the BMD of femoral neck decreased with the increase of blood manganese in 50-59 and 60-69 years [(β coefficient -0.0581; 95%CI -0.1151, -0.0011; P 0.046); (β coefficient -0.0561. 95% CI -0.1104, -0.0018; P 0.043)], and there is a negative correlation between BMC and blood manganese.    In the spinal region, gender stratified results have showed that only the total spinal BMC is significantly correlated with the blood manganese, and no significant correlations are found between the total spinal BMD and the blood manganese in all gender groups. Results stratified by race have showed that there is no significant correlation between BMD/BMC and blood manganese in all races. After stratified by age, BMD/BMC is negatively correlated with blood manganese in the total spine at 50-59 years of age [(β coefficient -0.0978; 95%CI -0.1697, -0.0259; P 0.008); (β coefficient -0.1021; 95% CI -0.1717, -0.0325; P 0.004)].
In the total body region, after stratified by sex, BMD decreased significantly with blood manganese increasing in males (β coefficient -0.1455; 95% CI -0.1976, -0.0934; P <0.001), while total body BMC decreased with blood manganese increasing in all gender groups (β coefficient < 0, P <0.05). Results stratified by race have showed that there is no significant

Discussion
The incidence of osteoporosis is rising, with the increasing longevity of global population. According to International Commission on Radiation Protection (ICRP), about 40% of body manganese accumulates in bones [17]. Thus, we hypothesized that blood manganese might somehow be associated with BMD/BMC. In contrast to previous studies, after adjusting for confounders, we found that BMD in the femoral neck and total body is negatively associated with blood manganese, while BMD in the spine is not significantly negatively associated with blood manganese. The BMC of femur, spine and total body has showed significant negative correlation with blood manganese. Subgroup analysis have showed that for BMD, blood manganese is negatively correlated with femur neck BMD in females aged between 50-70 years, spinal BMD in 50-60 years old, and total body BMD in males and most ages of 18-60 years old. For BMC, blood manganese is inversely associated with femur neck BMC in males or females, 50-70 years of age, spinal BMC in males, 50-60 years of age, and total body BMC in males or females, Mexican Americans, and 18-60 years of age. Multiple linear regression analysis and subgroup analysis have showed that the relationship between blood manganese and BMD has mainly been found on the femoral neck and total body, but not the spine. This may be attributed to the different structure of the femur and spine, as previous studies shown, the trabecular bone of the lumbar spine is dominant compared to the femoral neck, which contains a higher proportion of cortical bone. Although trabecular bone makes up only 20% of bone mass in healthy adult bones, it has a greater surface area and remodeling rate than cortical bone [18,19]. However, due to the lack of research on the specific mechanism of blood manganese in bone metabolism, further basic research is necessary. Interestingly, it is noteworthy that no matter in femoral neck, spine or total body, blood manganese concentration is significantly negatively correlated with BMD/BMC in people between 50-60 years old, indicating that blood manganese concentration has certain predictive value for bone mineral density as well as bone mass in those between 50-60 years old. The explanation for this phenomenon is unclear, however, owing to changes in physical function, Table 6. Association of blood manganese with total body BMD/BMC, stratified by sex, race/ethnicity and age.

Total body BMD(g/cm 2 ) Total body BMC(g)
β (95% CI) P value β (95% CI) P value age may be a common factor influencing an individual's susceptibility to manganese toxicity. In addition, our study has found that in the femoral neck, blood manganese is significantly negatively correlated with BMD in female instead of male, while in the total body, blood manganese is significantly negatively correlated with BMD in male rather than female. On the one way, it may account for this difference that the femur DEXA data focused on people over 40 years of age while the DEXA data on total body are concentrated in people aged 8-59, and the data under 18 years old are excluded in the study. On the other way, we speculate that different sex hormones levels may have different function on the BMD in femoral neck or total body. Previous studies have shown that most changes in BMD, especially in the hip, can be attributed to BMC variation, and both are considered to be important determinants of osteoporotic fractures [20]. In our study, blood manganese is significantly negatively correlated with BMC in most regions of femur, spine and total body. It is consistent with previous finding that bone weight reduction was observed in rats with long-term oral manganese intake [21]. However, the association between blood manganese level and bone loss remained debatable, and some previous epidemiological studies have shown mixed findings. A study determining the effect of manganese (Mn) supplementation on BMD and bone metabolic parameters in ovariectomized Sprague-Dawley rats found that manganese supplementation improved mineral density and serum osteocalcin in the spine and femur of rats [22]. Another prospective cross-sectional study of 41 untreated postmenopausal women showed a significant positive association between serum manganese levels and lumbar or femoral BMD [23]. Odabasi et al., in a casecontrol study of 138 postmenopausal women, reported that there were no significant differences in blood manganese levels between postmenopausal women with osteoporosis and postmenopausal women without osteoporosis. The median blood manganese concentration was 14.76 ng/mL in the osteoporosis group and 15.54 ng/mL in the control group [24]. Wang et al found no correlation between plasma manganese and bone mineral density in an epidemiological study of 91 elderly men over 50 years old in Beijing [25]. However, in a cross-sectional study of 304 retired workers with occupational manganese exposure, aiming to investigate the relationship between long-term occupational manganese exposure and bone quality, female participants in the highest manganese exposure group were significantly lower than the female control group in terms of stiffness index (SI) and T-score levels. This suggests that women with occupational manganese exposure may have a higher risk of osteoporosis [14]. Different sample sizes, inclusion and exclusion criteria, duration of follow-up, and different levels of manganese exposure may lead to different results between studies. These results indicate that the relationship between blood manganese and bone mineral density is still uncertain, and further basic studies are needed to explore the relationship between manganese and bone metabolism.

Men
Our study shows that blood manganese levels are associated with bone mineral density of femur and total body, and high blood manganese levels may affect bone metabolism. Bone is one of the body's main organs for long-term storage of manganese, and according to the International Commission on Radiation Protection (ICRP), about 40% of body manganese accumulates in bones [17]. Evidence from animal study suggested that Mn accumulated in rat bones with an average elimination half-life of 143 days, equivalent to approximately 8.5 years in human bones [21]. Thus, bone may also be an ideal organ exploring toxicity which caused by long-term manganese exposure [14]. It has been previously reported that excessive manganese may be neurotoxic to humans, affecting specific areas of the central nervous system and producing irreversible neurological symptoms [26]. Occupational exposure concentrations of airborne manganese in the range of 0.1-1.27 mg/m 3 indicate blood levels of 10.3-12.5 ug/ L [27]. In patients with clinical signs and symptoms of manganese poisoning, blood manganese concentration varies between 4 and 40 ug/L [21,[28][29][30]. Studies have shown that dietary manganese deficiency and excessive manganese exposure can increase the production of reactive oxygen species (ROS), and abnormal accumulation of excessive ROS will exacerbate oxidative stress and inflammation, which is crucial in the pathogenesis of metabolic diseases (31). Recent studies [31,32] have found that during osteoblast differentiation, mitochondrial biogenesis, mitochondrial function (especially the activity of complex I in mitochondrial ECT) and ATP content are significantly increased, while excessive manganese accumulation in mitochondria inhibits mitochondrial complex I and II respiration and induces permeability transition [33][34][35][36]. It may disrupt mitochondrial homeostasis and lead to mitochondrial dysfunction, ultimately adversely affecting BMD. In addition, proposed in recent years, mitophagy has been reported that it improves tissue homeostasis via recovering energy by limiting the energy demands of ineffective organelles, reducing intracellular ROS produced by impaired mitochondria, and generating ATP during degradation under physiological or pathological conditions, while the disorder of mitophagy damages cells energy metabolism and physiological function, and breaks mitochondrial homeostasis [31,37]. Thus, we speculate that excess manganese may also adversely affect bone metabolism through the mitophagy pathway, and the mechanism of this still needs further studied.
Significantly higher blood Mn levels have been revealed in women of US residents, Canadians, Koreans, Chinese general population, and Italians [14,[38][39][40][41][42]. Mining and iron as well as steel production are considered as occupational sources of manganese exposure. Humans are more likely to be exposed to manganese through diet. Nuts, grain products, chocolate, crustaceans, legumes, mollusks, fresh fruits and tea are considered as rich sources of manganese [43]. In the United States, the recommended daily intake is 1.8 mg/day for women and 2.3 mg/ day for men [44]. Excessive intake of manganese has been reported to be associated with cognitive impairment in human [45,46]. According to the National Institutes of Health, consuming more than 11 milligrams a day may have bad effects and the typical non-vegetarian Western diets provide 3-7 mg of manganese per day [47]. Previous studies have suggested that routine supplementation of manganese and other metals is generally unnecessary and such excessive supplementation may be harmful [44]. In the current study, we reported a negative association between blood manganese in adults selected from NHANES in 2013-2014 and 2017-2018 and femur and total body BMD/BMC. As the results showed, manganese intake may remain as an important public health issue. Some multivitamins typically contain 2-4 mg of additional manganese, and those with long-term intake, especially older adults, need to pay extra attention to their long-term risks and benefits. While in Europe, the European Food Safety Agency (EFSA), considering lacking of the sufficient and definitive data concerning humans, has neither established any tolerable upper intake Level (UL), nor an average requirement (Average Requirement, AR) and a reference intake for the population (population reference intake, PRI) for Mn [48,49]. The results of our study may provide some references for the establishment of AR, PRI and UL of Mn. In addition, regular dual-energy radiographs of the femur or total body are recommended for people exposed to manganese, including those living near factories where manganese is utilized in production, especially women, to be aware of the risk of osteoporosis.
This study has several strengths for exploring the relationship between blood manganese status and BMD/BMC in different populations. (i) Based on data from the NHANES database, this study has a large sample size and sufficient clinical information. (ii) After adequately controlling for confounding factors, this study estimated the difference in the association between blood manganese status and BMD/BMC in diverse populations by stratifying age, sex and race. (iii) This is the first study that found decreased femoral neck BMC and BMD in female participants aged 50-70 had higher blood manganese levels, which suggests that blood manganese might have potential value in predicting osteopenia, osteoporosis and fragility fractures.
In addition, this study still has some limitations. (i) As a cross-sectional study, the causal relationship between Mn and BMD/BMC could be bidirectional. Nevertheless, great attention should be paid to the influence of blood manganese level in daily diet and life. More clinical trial and basic researches about the mechanism should be performed in the further. (ii) The total body BMD and BMC in the NHANES database only included data from samples aged 8-59 years (we excluded data under 18 years old), while the BMD and BMC of femur and spine only included data over 40 years old, which could not properly represent the American population over 18 years old. (iii) The study only enrolled the participants in the United States, so the results may not accurately apply to other countries or regions such as Asia and Africa. (iv) Residual or unmeasured confounders are possible despite adjustment for potential known confounders.

Conclusion
This nationwide cross-sectional study has showed a negative association between blood manganese levels and femur as well as total body BMD/BMC, especially femoral neck BMD in women aged 50-70 years. People exposed to manganese should be aware of the increased risk of osteopenia or osteoporosis. Besides, due to the lack of available data, there are no definite values for the tolerable upper intake level (UL), average requirement (AR) and population reference intake (PRI) of manganese. The results of our study may provide some references for the establishment of AR, PRI and UL of Mn.
Supporting information S1 Table. Related demographic characteristics of participants included in NHANES from 2013 to 2014 and 2017 to 2018, based on blood manganese quartiles. Means with standard deviation (SD) were used for continuous characteristic variables and categorical variables were expressed as frequencies. Differences in categorical variables between exposed groups were analyzed by Pearson's chi-square tests. One-way ANOVA was used to analyze the differences of continuous variables between groups. (DOCX) S2 Table. Blood manganese and area, BMC, BMD based on blood manganese quartiles. Data are expressed as means (standard deviation, SD). BMC, bone mineral content, BMD, bone mineral density, Area (cm 2 ), BMC (g), BMD (g/cm 2 ). (DOCX)